Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy

Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3 h after intravenous injection and rapid clearance from normal tissues within 24 h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3 h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use.     

Human acidic mammalian chitinase as a novel target for anti-asthma drug design using in silico screening

Human acidic mammalian chitinase (hAMCase) was recently shown to be involved in the development of asthma, suggesting a possible application for hAMCase inhibitors as novel therapeutic agents for asthma. We therefore initiated drug discovery research into hAMCase using a combination of in silico methodologies and a hAMCase assay system. We first selected 23 candidate hAMCase inhibitors from a database of four million compounds using a multistep screening system combining Tripos Topomer Search technology, a docking calculation and two-dimensional molecular similarity analysis. We then measured hAMCase inhibitory activity of the selected compounds and identified seven compounds with IC₅₀ values ?100 μM. A model describing the binding modes of these hit compounds to hAMCase was constructed, and we discuss the structure–activity relationships of the compounds we identified, suggested by the model and the actual inhibitory activities of the compounds.     

Diagnostic value of urine erythrocyte morphology in the detection of glomerular disease in SurePath™ liquid‐based cytology compared with fresh urine sediment examination

H. Ohsaki, T. Hirouchi, N. Hayashi, E. Okanoue, M. Ohara, N. Kuroda, E. Hirakawa and Y. Norimatsu
Diagnostic value of urine erythrocyte morphology in the detection of glomerular disease in SurePath? liquid‐based cytology compared with fresh urine sediment examination Objective: To assess whether the morphology of urine erythrocytes can be an effective tool for distinguishing glomerular disease from lower urinary tract disease in SurePath^? liquid‐based cytology (SP‐LBC). Methods: We examined four morphological parameters of erythrocytes: (1) irregular erythrocytes (of all types including fragmented forms) comprising greater than or equal to 20% of erythrocytes; (2) uniform erythrocytes (>80%); (3) doughnut or target‐like shaped (D/T) erythrocytes (≥1%); and (4) acanthocytes (≥1%) in glomerular disease (n = 32) and lower urinary tract disease (n = 20) with SP‐LBC slides in cases that had also been assessed by fresh urine sediment examination. Results: Sensitivity of D/T erythrocytes and acanthocytes (dysmorphic erythrocytes) for glomerular disease were 100% and 87.5%, respectively, with urine sediment examination, and 81.3% and 46.9%, respectively, in SP‐LBC slides. Specificity was 100% for D/T erythrocytes and acanthocytes using either procedure. While irregular erythrocytes were specific for glomerular disease using urine sediment examination, they were seen in 70% of those with lower urinary tract disease using SP‐LBC slides as a result of the deformation of erythrocytes by the fixative. Conclusions: Although the sensitivity of D/T erythrocytes and acanthocytes for glomerular disease was lower in SP‐LBC slides than fresh urine sediment examination, their specificity was equally high. Therefore, urine erythrocyte morphology is useful in the detection of glomerular disease with the SP‐LBC slides. However, morphological features apart from D/T erythrocytes and acanthocytes are not useful in SP‐LBC slides.     

Components Contributing to the Improvement of Meat Taste during Storage

The changes in both taste and taste components of beef, pork, and chicken during storage were examined.

The brothy taste intensity of pork and chicken was significantly stronger after conditioning than before. On the other hand, for beef, there was no significant difference in the brothy taste intensity before or after conditioning. The analysis of major taste components showed that the levels of free amino acids in all meats were higher after conditioning than before. The differences in the levels of free amino acids before versus after conditioning were large in pork and chicken and very small in beef. Oligopeptide levels were lower in beef after conditioning than before, but they were higher in pork and chicken after conditioning than before. These results corresponded to results of the sensory evaluation studies described above, indicating that free amino acids and oligopeptides contributed to the improvement of meat taste during storage.     

Purification of Alkaline Proteinase from Aspergillus Candidus

An alkaline proteinase of Aspergillus Candidus was purified from wheat bran solid culture by batchwise treatment with Amberlite IRC–50 and sequential chromatography on DEAE-cellulose, hydroxylapatite and Sephadex G–100 gel. This purification results in a 18-fold increase of proteolytic activity and the enzyme preparation was homogeneous in sedimentation analysis of the ultracentrifuge and polyacrylamide gel disc electrophoresis. The molecular weight was estimated to be about 23,000 by gel glltration and 22,000 by calculation from the amino acid composition. The enzyme consisted of Lys₁₄, His₄, Arg₃, Asp₂₅, Thr₁₅, Ser₂₃, Glu₁₅, Pro₇, Gly₂₂, Ala₂₄, Met₂, Val₁₆, Ile₁₁, Leu₁₀, Tyr₆, Phe₇, Trp₂ and amide ammonia₁₄ and did not contain cysteine or cystine.     

Enzymatic Properties of Alkaline Proteinase from Aspergillus candidus

Some enzymatic properties were examined with the purified alkaline proteinase from Aspergillus candidus. The isoelectric point was determined to be 4.9 by polyacrylamide gel disc electrofocusing. The optimum pH for milk casein was around 11.0 to 11.5 at 30°C. The maximum activity was found at 47°C at pH 7.0 for 10 min. The enzyme was stable between pH 5.0 and 9.0 at 30°C and most stable at pH 6.0 at 50°C. The enzyme activity over 95% remained at 40°C, but was almost completely lost at 60°C for 10 min. Calcium ions protected the enzyme from heat denaturation to some extent. No metal ions examined showed stimulatory effect and Hg²⁺ ions inhibited the enzyme. The enzyme was also inhibited by potato inhibitor and diisopropylphosphorofluoridate, but not by metal chelating agent or sulfhydryl reagents. A. candidus alkaline proteinase exhibited immunological cross-reacting properties similar to those of alkaline proteinases of A. sojae and A. oryzae.     

Effect of Cathepsin D Treatment on ATPase Activity of Rabbit Myofibril

The effect of cathepsin D and pepsin treatment on rabbit myofibril was studied by measuring the amount of proteolytic products and Mg-enhanced ATPase activity.

When myofibril was treated with cathepsin D at 3°C and pH 5.0 or 5.5, a little but detectable amount of nonprotein nitrogenous compounds was released. However, there was no change in ATPase activity of myofibril, though treated with cathepsin D of higher units than assumed to be in muscle.

When myofibril was treated with pepsin under the same condition as used above, there was an increase in KCl-concentration dependence of ATPase activity followed by a decrease in the maximal value of ATPase activity.

From the present results, it was concluded that cathepsin D might not take a main role on the post-mortem degradation of myofibril.     

Successive Isolation of Proteinase Hyperproductive Mutants of Aspergillus sojae

Hydrochloric acid treatment of methyl 3-(4-isobutylphenyl)-3-methylglycidate and methyl 2-hydroxy-3-(4-isobutylphenyl)-3-butenoate, a rearrangement product of the former, in acetic acid gave 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-isobutylphenyl)-pro-panal. The same treatment of 2-hydroxy-3-(4-isobutylphenyl)-3-butenoic acid gave 2-(4-isobutylphenyl)-propanal. Both 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-iso-butylphenyl)-propanal were oxidized to 2-(4-isobutylphenyl)-propionic acid.     

Properties of Nucleoside Phosphorylase from Enterobacter aerogenes

The properties of uridine Phosphorylase (UPase) and purine nucleoside Phosphorylase (PNPase) at high temperature were investigated. Both enzymes were found to be distributed in a wide range of bacteria and were partially purified from Enterobacter aerogenes AJ 11125 by heat treatment, ammonium sulfate fractionation and column chromatographies onDEAE-cellulose and Sephadex G-150. The UPase was purified 109-fold, and it showed an optimum pH of 8.5 and optimum temperature of 65°C, and activity toward uridine, 2′-deoxyuridine, thymidine and uracil arabinoside but not cytidine. The Km values of UPase for uridine were 0.7 mm at 40°C and 1.8 mm at 60°C. The PNPase was purified 83-fold, and it showed an optimum pH of 6.8 and optimum temperature of 60°C, and significant activity toward purine arabinosides as well as purine ribosides. The Km values of PNPase for inosine were 0.8 mm at 40°C and 2.2 mm at 60°C.